Method for detoxifying a biological tissue

ABSTRACT

Biological tissues may be prepared for use in biological prostheses. The biological tissue may be fixated with glutaraldehyde and may be subjected to successive treatment of the tissue with a solution containing taurine to neutralize excess aldehyde groups that remain free after fixation.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/280,804, filed May 19, 2014, which is a continuation of U.S.application Ser. No. 13/154,856, filed Jun. 7, 2011, which claimspriority to Italian Patent Application No. IT TO2010A000486 filed Jun.9, 2010, and claims priority to European Application No. 11167681, filedMay 26, 2011, all of which are incorporated by reference herein in theirentirety.

TECHNICAL FIELD

The present invention is generally directed to a method for detoxifyingbiological tissue for use in biological prostheses such as prostheticvalves and more particularly to a method for detoxifying biologicaltissue for use in prosthetic cardiac valves.

BACKGROUND

Biological prostheses are medical devices that utilize animal tissues.Examples of suitable animal tissues include bovine, porcine, ovine andequine. Depending on the various medical uses, the biological tissueinclude cardiac valves, pericardium, tendons, ligaments, dura mater,skin, veins, etc.

The animal tissues used in biological prostheses are formed primarily ofcollagen, a protein with a structural unit represented by threepolypeptide chains that associate to form a triple helix. Collagenmolecules assemble to form microfibrils that in turn assemble to formfibrils that, arranged in corrugated or parallel bundles, give rise totrue collagen fibers. Such tissues have good resistance to traction andare flexible but substantially inextensible.

Animal tissues used in biological prostheses are first subjected tonumerous washings to eliminate traces of blood and a careful removal ofadipose and ligamentous parts. However, cells or cellular residues fromthe animal donor can remain trapped in the structure of the tissueitself. As a result it is possible that the immune system of the hostgives rise to a rejection phenomenon that can lead to the destruction ofthe tissue constituting the biological prosthesis.

An additional problem is degradation of the collagenous biologicaltissue once implanted in the host organism. For this reason, thebiological tissues are subjected to a fixation treatment with the aim ofprotecting the tissue from such degradation phenomena and contributingto preventing the above-mentioned rejection phenomenon.

Among the substances used for the fixation of biological tissues, themost common is glutaraldehyde. This bifunctional molecule, carrying twoaldehyde groups, is capable of stably binding together free amino groupsof the amino acids that constitute the polypeptide chains both withinone collagen molecule and between adjacent collagen molecules. In thisway glutaraldehyde forms intra-chain and inter-chain bridge structures,causing the cross linking of biological tissue. Such cross linkingprotects the tissue from degradation by the host and confers favorablemechanical properties such as for example a better resistance totraction with respect to untreated tissue.

Glutaraldehyde is a highly bactericidal and virucidal substance;therefore, in addition to cross linking the tissue, the fixation stepalso provides at least a partial sterilization.

In addition, glutaraldehyde is capable of binding to the free aminoresidues of the membrane proteins of the cellular components stillpresent, masking their antigenic potential and impeding immuneactivation phenomena and rejection by the host.

In spite of widespread use, glutaraldehyde has a disadvantage of beingone of the factors that favors pathological calcification of implantedtissues. The calcium, present in the bodily fluids of the host organism,accumulates in proteinaceous tissue giving rise, for example in the caseof biological cardiac valves, to a process that may represent one of theprinciple causes of valve failure. The calcium deposits can reduce theflexibility of the portion of biological tissue constituting the valve(or the so-called valve leaflets or cusps) and lead to laceration of thetissue itself, causing a partial or total loss of valve function.

The mechanism responsible for calcification is not yet completely knownand is attributed to numerous factors; however, it is known that afterglutaraldehyde fixation, aldehyde groups remaining free on the tissuecan create binding sites for calcium.

In addition, the toxicity of such aldehyde residues can cause localinflammatory phenomena that lead to the necrosis of host cells.Destruction of the dead cells gives rise to cellular debris that, inturn, can constitute binding cites for calcium. Several types ofmolecules capable of neutralizing the aldehyde residues remaining freeafter the fixation process have been used to limit the process of tissuecalcification. For example, the use of amino acids has been shown tohave an anti calcification effect; in particular, U.S. Pat. No.5,873,812 describes the use of amino carboxylic acids, such as forexample homocysteic acid, in the preparation of aldehyde-fixedbiological tissues. However, this method only partially neutralizes freealdehyde groups and thus does not resolve the problem.

SUMMARY

The present invention is directed to improved, more efficacioussolutions that limit the calcification of biological tissues afterimplantation in the host.

In some embodiments, the present invention is directed to a method fortreating a biological tissue for biological prostheses. The methodincludes a tissue fixation step and a detoxification step.

In some embodiments, the method for treating a biological tissueincludes fixation of the biological tissue via a treatment with aglutaraldehyde solution and detoxifying the fixed biological tissues viaa treatment with a taurine solution at a temperature that is in therange of about 30 to about 45 degrees Celsius.

In some embodiments, the results presented below show that the methoddescribed herein strongly reduces the number of free aldehyde groupspresent on fixed tissue, presenting an clear advantage with respect tomethods that envision the detoxification of tissue by means of immersionin a solution containing homocysteic acid.

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described in detail, by way of non-limitingexample only, with reference to the attached figures, in which:

FIG. 1A shows fuchsine staining of a control sample fixed but notdetoxified.

FIG. 1B shows fuchsine staining of a sample that was fixed anddetoxified according to the known art using a homocysteic acid solution.

FIG. 1C shows fuchsine staining of a sample that was fixed anddetoxified with a taurine solution in accordance with embodiments of thepresent invention.

FIG. 2A fuchsine staining of a control sample fixed but not detoxified.

FIG. 2B shows fuchsine staining of a sample that was fixed anddetoxified with a taurine solution at room temperature.

FIG. 2C shows fuchsine staining of a sample that was fixed anddetoxified with a taurine solution at 40 degrees Celsius.

FIG. 2D shows fuchsine staining of a sample that was fixed anddetoxified with a taurine solution at 50 degrees Celsius.

DETAILED DESCRIPTION

The invention will now be described in detail, by way of non-limitingexample only, with reference to forming biological prosthetic cardiacvalves. It is clear that the procedure described herein can be used forthe detoxification of any other biological tissue destined for formingother biological prostheses that use, for example tendons, ligaments,dura mater, skin, veins, etc.

In the following description, numerous specific details are given toprovide a thorough understanding of the embodiments. The embodiments canbe practiced without one or more of the specific details, or with othermethods, components, materials, etc. In other instances, well-knownstructures, materials or operations are not shown or described in detailto avoid obscuring certain aspects of the embodiments.

Reference throughout the present specification to “one embodiment” or“an embodiment” means that a particular feature, structure orcharacteristic described in connection with the embodiment is includedin at least one embodiment. Thus, the phrase “in one embodiment” or “inan embodiment” in various places throughout the present specificationare not necessarily all referring to the same embodiment. Furthermore,the details of features, structures, or characteristics may be combinedin any suitable manner in one or more embodiments.

The headings provided herein are for convenience only and do notinterpret the scope or meaning of the embodiments.

The present invention concerns a method for treating a biological tissuethat includes two steps:

-   -   i) Fixation of the biological tissue via treatment with a        solution containing glutaraldehyde, and    -   ii) Detoxification of the previously fixed biological tissue via        treatment with a solution containing taurine.

In some embodiments, the fixation step includes immersion of the tissuein a solution containing glutaraldehyde for a period that varies betweenone minute and 3 days and a maximum of 13 days. Fixation cross links thetissue, which confers it with resistance to degradation and favorablemechanical properties.

In some embodiments, the detoxification step includes immersion of thepreviously fixed tissue in a solution containing taurine for a periodthat varies in a range from one minute to a few hours and a maximum ofseveral days.

Taurine (also known as aminoethanesulfonic acid) has an amine group(NH₂) available to bond with the aldehyde groups of glutaraldehyde thatremain free after the tissue fixation step. In particular, the presentinventors discovered that the use of taurine in the detoxification stepin place of homocysteic acid provides greater efficacy for neutralizingthe aldehyde groups that remain free after glutaraldehyde fixation,therefore reducing in a highly efficacious way the number of sites ofbinding and accumulation of calcium on the biological tissue.

Biological tissues to be used for biological prosthetic cardiac valves,or bovine pericardium, equine or possibly pericardium of other animalspecies or porcine valves are harvested from authorized abattoirsand—transported to the laboratory—immersed in saline solution maintainedon ice to avoid damage to the tissue before use.

The tissues are washed with saline solution to eliminate possible tracesof blood, separated from possible adherent ligamentous and adiposeparts, and then carefully selected on the basis of thickness and on thebasis of the absence of evident defects such as dishomogeneity ofthickness, presence of cuts, abrasions, etc.

The tissues are initially prefixed in a glutaraldehyde solution at roomtemperature in a volume/volume concentration that is in the range of0.05% to 0.30% glutaraldehyde in phosphate buffer at a pH of 7.4 for aperiod of time that ranges from 3 to 13 hours. In some embodiments, theglutaraldehyde solution includes 0.20% glutaraldehyde

After the prefixation step, in some embodiments, comes a step of cuttingand shaping the tissue—according to known art techniques—to form, forexample, cardiac valves. This step of shaping the tissue is irrelevantto the aims of the method object of the present description.

The method of fixation follows and is carried out by immersing thetissue in a solution containing glutaraldehyde at a concentration in therange 0.30% to 1.00%. In some embodiments, the solution containsglutaraldehyde at a concentration of 0.5%.

In some embodiments, the solution containing glutaraldehyde is anaqueous solution including a buffer selected from phosphate, citrate,acetate, HEPES, and borate. In some embodiments, the buffer isphosphate.

The pH of the glutaraldehyde-based solution is in the range of 5 to 8.In some embodiments, the pH of the glutaraldehyde-based solution is 7.4.

The fixing method is conducted at a temperature in the range 4° C. to30° C. In some embodiments, the fixing method is conducted at roomtemperature (20° C.). The period of exposure of the tissue to thesolution containing glutaraldehyde can vary in the range 1 to 20 days.In some embodiments, the exposure time is in the range of 3 to 13 days.

After the fixation step, the tissue is washed to remove residualglutaraldehyde that is not conjugated to the tissue. In someembodiments, the wash solution is a saline solution or a phosphatebuffer pH 7-7.4 and is changed three times. Washing is performed for aperiod comprised in the range 30 minutes to 6 hours, with gentleagitation, at room temperature.

The fixed tissue is then detoxified using an aqueous solution containingtaurine at a concentration w/v comprised in the range of 0.10% up tosaturation of the solution. In some embodiments, the aqueous solutionincludes taurine at a w/v concentration of 0.20% to 1.00%. In someembodiments, the aqueous solution includes taurine at a concentration of0.70% w/v.

In some embodiments, the aqueous taurine solution contains a bufferselected from phosphate, citrate, acetate, HEPES(4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid), and borate. Insome embodiments, the buffer is phosphate. In some embodiments, the pHof the solution is in the range of 4 to 9. In some embodiments, the pHof the solution is 5 to 8. In some embodiments, the pH of the solutionis 7.

In some embodiments, the detoxification step is conducted at atemperature in the range from room temperature (20° C.) to 50° C. Insome embodiments, the detoxification step is conducted at a temperaturethat is in the range of 30° C. to 45° C. In some embodiments, thedetoxification step takes place at a temperature of 40° C. In someembodiments, the period of immersion of the tissue in thetaurine-containing solution may vary in the range from 2 to 96 hours. Insome embodiments, the immersion period may be in the range of 12 to 48hours. In some embodiments, the immersion period is 24 hours.

At the end of the detoxification step, the detoxified tissue issubjected to washing at room temperature, in phosphate buffer at pH 7,for about three hours, changing the wash solution three times.

The detoxified tissue is finally transferred to a conservation solutionwithout aldehydes in phosphate buffer pH 7 containing preservatives suchas parabens.

The efficacy of the method described herein, and therefore theoccurrence of the reaction between the amino groups of the detoxifyingmolecule and the aldehyde groups present on the fixed tissue, isevaluated by staining of the aldehyde groups remaining free. The greaterthe staining intensity, the more numerous are the free aldehyde groupsand, on the contrary, the weaker (or absent) the staining, the lessnumerous (or absent) are the free aldehyde groups present on thedetoxified tissue. An example of a stain advantageously useful for suchdetermination is fuchsine.

The results presented below demonstrate that taurine binds the freealdehyde groups present on fixed tissue more efficaciously thanhomocysteic acid. In addition, the present inventors have observed thatbetter results are obtained by performing the detoxification step at atemperature above room temperature, that is, the number of free aldehydegroups present on the fixed tissue are greatly reduced.

The various preferred embodiments of the present invention will bedescribed in detail below.

Materials and Methods Harvesting and Fixation of Biological Tissue

The biological tissue, which were pieces of bovine pericardium harvestedfrom authorized abattoirs, was placed in a saline solution maintained onice and transported to the laboratory.

The tissues were washed with saline solution to eliminate possibletraces of blood, separated from the any adherent ligamentous and adiposeparts and were selected on the basis of the correct thickness and theabsence of defects, such as dishomogeneity in thickness, presence ofvascularizations, cuts, abrasions, etc.

The tissues were prefixed in a glutaraldehyde solution at 0.20% v/v inphosphate buffer pH 7.4, for a time variable in the range of 3 to 13hours at room temperature.

After the prefixation step, the tissues were cut and shaped according toknown art techniques—for making, for example, cardiac valves. Next thetissues were fixed for a period of time in the range 3 to 13 days atroom temperature, in an aqueous solution of 0.50% v/v glutaraldehyde inphosphate buffer pH 7.4.

Detoxification of Tissue

The tissue to be detoxified was washed, to remove residualglutaraldehyde solution, in saline solution or in phosphate buffer pH7.4 or pH 7, for about three hours, with gentle agitation, at roomtemperature. The wash solution was changed three times, using about 300ml of solution each time for each piece of tissue with dimensions ofabout 10×5 cm.

The fixed solution was then detoxified using an aqueous solutioncontaining taurine at a concentration (w/v) of about 0.70% in phosphatebuffer pH 7. The detoxification step was conducted at room temperature(20° C.), 40° C. and 50° C., for a period of time of 24 hours, usingabout 200 ml of solution for each tissue.

At the end of the detoxification step, the detoxified tissue was washedat room temperature, in phosphate buffer at pH 7, for about three hours,changing the wash solution three times.

Finally, the detoxified tissue was transferred to a conservationsolution without aldehydes containing preservatives, preferablyparabens, in phosphate buffer pH 7.

With the aim of demonstrating the better efficacy of the detoxifyingtreatment with taurine compared to the known art, the present inventorshave semi-quantitatively determined the number of free aldehyde groupsin three samples of biological tissue treated as follows:

a first control sample (FIG. 1A) including biological tissue fixed withglutaraldehyde but not detoxified;

a second control sample (FIG. 1B) fixed with glutaraldehyde anddetoxified with a solution containing homocysteic acid at aconcentration (w/v) of 1.00% in phosphate buffer pH 7;

a third sample (FIG. 1C) fixed with glutaraldehyde and detoxified with asolution containing taurine at a concentration (w/v) equal to 0.70% inphosphate buffer pH 7.

The tissue samples were immersed in the detoxifying solutions for about24 hours at room temperature with gentle agitation.

To verify the efficacy of the taurine detoxifying treatment withvariation of the detoxifying solution temperature, the present inventorssemi-quantitatively determined the number of free aldehyde groups inbiological tissues treated as follows:

a first control sample (FIG. 2A) including biological tissue fixed withglutaraldehyde but not detoxified;

a second sample (FIG. 2B) fixed with glutaraldehyde and detoxified witha solution containing taurine at a temperature of 20° C.;

a third sample (FIG. 2C) fixed with glutaraldehyde and detoxified with asolution containing taurine at a temperature of 40° C.; and

a fourth sample (FIG. 2D) fixed with glutaraldehyde and detoxified witha solution containing taurine at a temperature of 50° C.

The samples were immersed in the detoxifying solution for about 24 hourswith gentle agitation both at room temperature and at 40° C.; the fourthsample detoxified at 50° C. was maintained immersed for 7-8 hours withgentle agitation.

About 200 ml of detoxifying solution were used for each sample.

At the end of the treatment all samples were washed at room temperaturein phosphate buffer at pH 7, for about three hours, changing the washsolution three times and using about 300 ml of solution for each sampleat each change.

All samples were then transferred to a phosphate buffer solution pH 7containing preservatives such as parabens.

Staining of the Tissue with Fuchsine

Tissue staining to detect free aldehyde groups uses an acidic solutionof rosaniline hydrochloride (fuchsine). The staining takes advantage ofthe formation of bonds between the NH₂ groups of the dye and the freealdehyde groups on the tissue.

At first, the solution is colorless but in the presence of free aldehydegroups a violet color develops. This is a qualitative evaluation of theavailability of free aldehyde groups, after the various treatments.

The samples to stain were cut to obtain cards with dimensions of about1.5×1.5 cm and successively immersed in the staining solution, about 10ml, each card in a separate test tube.

The staining solution was 1.00% rosaniline hydrochloride, 4.00% sodiummetabisulfite in 0.25 M hydrochloric acid. The samples remained immersedin the stain for 5 minutes at room temperature, with gentle agitation.

Each sample was then transferred to a solution obtained mixing 8 gr ofNa₂SO₃ and 30 ml of 37% hydrochloric acid, brought to one liter withdemineralized water. The samples remained immersed for 10 minutes inthis wash solution, with mild agitation.

The samples were then subjected to 2 successive 10 minute washings, withmild agitation in a wash solution including 700 ml of ethanol and 30 mlof 37% hydrochloric acid, brought to one liter with demineralized water.

The washings removed stain that was non-specifically bound to thetissue. About 20 ml of wash solution was used at each change.

When finished the samples were transferred to phosphate buffer pH 7 andphotographed to document the different staining.

Reflectance Spectroscopy

The stained samples were subjected to reflectance spectroscopy toquantitatively evaluate the different chromatic characteristics of thefuchsine staining.

Reflectance spectroscopy is a technique for optical investigation basedon measurement of the spectral reflectance factor of the surface of asample as a function of the wavelength of incident radiation. Thereflectance parameter is expressed as the ratio of the intensity of thereflected radiation and the incident radiation, as a function ofwavelength.

Reflectance measurements were carried out at a wavelength of 570 nmusing a Perkin Elmer Lambda 35 spectrophotometer with a sphericalintegrator.

On a scale of values, a lower reflectance value indicates a more intensesample staining and on the contrary a higher reflectance value indicatesa weak staining intensity.

Determination of the Contraction Temperature

The contraction temperature is an index of the level of cross linking ofthe fixed tissue and was determined on disks of pericardium of about 5mm diameter, using a scanning differential calorimeter (DSC)Q100 TAInstruments with the following parameters:

nitrogen flow of 50 ml/min,

heating ramp of 5° C./min,

temperature interval between 65° C. and 95° C.

Results Detoxification

The efficacy of the method described herein, and therefore of thereaction that takes place between the amino groups of the detoxifyingmolecule and the aldehyde groups on the fixed tissue, is demonstrated bystaining the aldehyde groups remaining free with fuchsine; more intensestaining indicates numerous free aldehyde groups and, on the contrary,weaker or absent staining indicates few or no free aldehyde groups onthe detoxified tissue.

FIGS. 1A-1C show a different intensity of staining of a sampledetoxified with a solution containing homocysteic acid, sample B, and ofa sample detoxified with a solution containing taurine, sample C. Asample not detoxified, sample A, has an intense purple color. Theintensity of staining of sample B is less than that of the controlsample, but the staining of sample C is very weak if not absent.

The number of free aldehyde groups present on the sample exposed to asolution containing taurine is therefore decidedly lower than that of asample not detoxified after fixation. Furthermore, treatment withtaurine is significantly more efficacious than that carried out withhomocysteic acid.

These observations are confirmed by the reflectance spectroscopicanalysis of the samples. The reflectance of each sample depends on thestaining intensity and by analysis carried out with a spectrophotometer,it is possible to associate a percent reflectance value to each sample.

A higher reflectance value is associated with weaker staining intensityand vice versa a lower reflectance value is related to a greaterstaining intensity.

As can be seen from the results reported in table 1, the reflectanceanalyzed at a wavelength of 570 nm and expressed as a percent shows avalue of 6.5 for the non detoxified sample shown in FIG. 1A, anintermediate value for the sample detoxified with homocysteic acid shownin FIG. 1B and a decidedly higher value for the sample detoxified with asolution containing taurine shown in FIG. 1C.

TABLE 1 Reflectance Staining % Sample observed at 570 nm A—nondetoxified Very intense 6.5 purple B—homocysteic acid Violet 9.2 roomtemperature C—taurine Pale violet 13 room temperature

These results confirm that treatment with taurine is more efficaciousthan treatment with homocysteic acid in neutralizing free aldehydegroups on the fixed tissue.

With reference to FIGS. 2A-2D, it can be seen how modifying thetemperature conditions under which the sample detoxification is carriedout with the solution containing taurine increases the efficacy of thetreatment. The staining intensity of the sample detoxified at 40° C.,sample C, is weaker with respect to that of the sample detoxified atroom temperature, sample B. Even more evident results are obtained byperforming the detoxifying treatment at a temperature of 50° C.; infact, sample D has a staining of decidedly lower intensity indicatingthat fewer free aldehyde groups remaining, and therefore sites forbinding and accumulation of calcium, with respect to the othertreatments.

The results of reflectance spectroscopic analysis for the samples shownin FIGS. 2A-2D are presented in table 2. The highest reflectance value,equal to 22, is from a sample having a staining intensity almost absent,that is the sample treated with taurine at 50° C. The non detoxifiedsample, which has very intense staining, has the lowest reflectancevalue. The sample treated with taurine at room temperature has a highervalue than the non detoxified control, but lower than the sample treatedwith taurine at 40° C.

TABLE 2 Staining Reflectance % Sample observed at 570 nm A—nondetoxified Very intense 6.5 purple B—taurine Pale violet 12 roomtemperature C—taurine 40° C. Pink 17 D—taurine 50° C. Completely 22colourless

Also in this case the reflectance spectroscopy results confirm thattreatment with taurine has a greater detoxifying capacity when conductedat a temperature higher than room temperature.

Contraction Temperature

To verify that the detoxifying treatment does not alter the crosslinking of the biological tissue obtained by means of immersion in asolution containing glutaraldehyde, the contraction temperature oftissue detoxified by means of treatment with a solution containingtaurine was compared to the contraction temperature of control tissuefixed and not detoxified.

The contraction temperature of tissues immersed in the solutioncontaining taurine at room temperature or at 40° C. areindistinguishable from the contraction temperature of the non detoxifiedtissue, that temperature being 85-86° C. Therefore, neither treatmenthas significant effects on the level of tissue cross linking.

The contraction temperature of tissue immersed in the solutioncontaining taurine at 50° C. remains the same as that of the nondetoxified tissue (85-86° C.) if the treatment period does not exceed7-8 hours.

Without prejudice to the underlying principle of the invention, thedetails and the embodiments may vary, even appreciably, with referenceto what has been described by way of example only, without departingfrom the scope of the invention as defined by the annexed claims.

We claim:
 1. A method of treating a biological tissue for use in a biological prosthesis, the method comprising the steps: treating the biological tissue with a fixation solution comprising glutaraldehyde at a concentration within the range from 0.3% v/v to 1.0% v/v, in a buffer at a pH from 5 to 8; and treating the biological tissue with a detoxification solution comprising taurine at a concentration within the range from 0.10% v/v to saturation in a buffer at a pH from 4 to 9, and at a temperature from 25° C. to 50° C.; wherein treating the biological tissue with the detoxification solution at a temperature range from 25° C. to 50° C. reduces calcification of the biological tissue when compared to treating a biological tissue at 20° C.
 2. The method according to claim 1, wherein the detoxification solution comprises the taurine at a concentration within the range of 0.20% v/v to 1.00% v/v.
 3. The method according to claim 1, wherein the detoxification solution comprises the taurine at a concentration of about 0.70% v/v.
 4. The method according to claim 1, wherein the detoxification solution buffer is selected from a phosphate buffer, a citrate buffer, an acetate buffer, a HEPES buffer, or a borate buffer.
 5. The method according to claim 1, wherein the detoxification solution buffer is a phosphate buffer.
 6. The method according to claim 1, wherein the detoxification solution has a pH in the range of 5 to
 8. 7. The method according to claim 1, wherein the detoxification solution has a pH of about
 7. 8. The method according to claim 1, wherein treating the biological tissue with a detoxification solution is carried out at a temperature of 40° C.
 9. The method according to claim 1, wherein treating the biological tissue with a detoxification solution is carried out for a period of 12 to 48 hours.
 10. The method according to claim 1, wherein the fixation solution comprises the glutaraldehyde at a concentration of about 0.5% v/v.
 11. A method of treating a biological tissue for use in a biological prosthesis, the method comprising the steps: fixing the biological tissue with a solution comprising glutaraldehyde at a concentration within the range from 0.3% v/v to 1.0% v/v, in a buffer at a pH from 5 to 8; and detoxifying the fixed biological tissue with a solution comprising taurine at a concentration within the range from 0.10% v/v to saturation in a buffer at a pH from 4 to 9, and at a temperature ranging from 20° C. to 50° C. wherein treatment of the biological tissue with the detoxification solution reduces calcification of the biological tissue when compared to treatment of a biological tissue using a detoxification solution comprising homocysteic acid.
 12. The method according to claim 11, wherein the fixing solution comprises glutaraldehyde at a concentration of about 0.5% v/v.
 13. The method according to claim 11, wherein the detoxification solution comprises the taurine at a concentration within the range of 0.20% v/v to 1.00% v/v.
 14. The method according to claim 11, wherein the detoxification solution comprises the taurine at a concentration of about 0.70% v/v.
 15. The method according to claim 11, wherein the detoxification step is carried out at a temperature of 20° C.
 16. The method according to claim 11, wherein the detoxification step is carried out at a temperature of 40° C.
 17. The method according to claim 11, wherein the detoxification step is carried out at a temperature of 50° C.
 18. The method according to claim 11, wherein the detoxification solution has a pH of about
 7. 